Process for the in vitro immunization of human splenocytes against tumor associated antigens

ABSTRACT

A process for the in vitro immunization of an immuno-competent splenocyte against an immunogen comprising (a) obtaining a first human splenocyte population, (b) fractionating the first population so that the first fraction is enriched with T-cells and a second fraction is enriched with B-cells, (c) mixing together the cells from the first and second fractions to form a second population having a T-helper cell to B-cell ratio of at least 0.4, and the percentage of T-suppressor cells is essentially unchanged from the first population, and (d) culturing the second population in a medium containing human serum, an immunogen and a lymphokine or lymphokines which induce proliferation and differentiation of T and B cells.

TECHNICAL FIELD

The present invention relates broadly to a process for the in vitroimmunization of human splenocytes against tumor associated antigens, andmore specifically, against bombesin.

BACKGROUND OF THE INVENTION

Techniques for the production of hybridomas and monoclonal antibodiesare well known. All such techniques require the immunization of alymphocyte against a preselected antigen. In many cases, especiallythose involving lymphocytes of non human origin, the immunization can becarried out by classical injection of the antigen into animmuno-competent host animal, followed eventually by sacrificing theanimal and removing the spleen which is a fertile source of immunelymphocytes.

With increasing interest in human monoclonal antibodies, it has becomenecessary to devise ways of immunizing human lymphocytes in-vitro.

Hoffmann discloses a process for producing human B-lymphocytescomprising culturing human B-lymphocytes in a tissue culture medium inthe presence of an antigen, helper signal producing agents comprisingmonocytes or monocyte conditioned medium containing Interleuken 1,helper T-lymphocytes or helper T-lymphocyte replacing factor, andhomologous human serum, followed by recovering the antibody producingcells from the medium. [U.S. Pat. No. 4,444,887 issued Apr. 24, 1984from an application filed Mar. 12, 1981.]

Cavagnaro and Osband (Biotechniques, 1, pp. 30-36 (1983)) disclose amethod for the in vitro primary immunization of human peripheral bloodmononuclear cells. The method requires three steps: (1) use ofautologous serum (autologous with cultures; (2) depletion of suppressorlymphocytes bearing H2 histamine receptors from human peripheral bloodmonocyte source; and (3) use of non-specific lymphocyte activator in theimmunizing cultures.

Erisman et al. describe the presence of bombesin in a human lungsmall-cell carcinoma grown in nude mice. Bombesin is a tetradecapeptideinitially isolated from frog skin. [P.N.A.S. (USA) 79, pp. 2379-2383,(1982).]

Moody et al. disclose the bombesin levels in small cell lung, but notother types of human cancer, are routinely elevated. [Peptides, Vol. 4pp. 683-686 (1983).]

SUMMARY OF THE INVENTION

The instant invention is a process (and product produced thereby) forthe in vitro immunization of an immuno-competent human splenocyteagainst a clinically relevant antigen. More specifically, the process ofthe instant invention comprises (a) obtaining a human splenocytepopulation (first population), (b) fractioning the first population sothat a first fraction is enriched with T-cells and a second fraction isenriched with B-cells (c) mixing together the cells from the first andsecond fractions to form a second population such that the secondpopulation has a T-helper cell to B-cell ratio of at least 0.4, and thepercentage of T-suppressor cells is substantially unchanged from thefirst population, and (d) culturing the second population in a mediumcontaining human serum, an immunogen, and a lymphokine(s) capable ofinducing proliferation and differentiation of T and B cells.

DETAILED DESCRIPTION

The human splenocytes utilized in the practice of the instant inventionare prepared by conventional techniques, one such technique beingdescribed in the example below. The splenocyte population so obtained isthen fractionated so that a first fraction is enriched with T-cells, anda second fraction is enriched with B-cells. By "enriched" it is meantthat the concentration of specific cell type is higher in thefractionated population than in the original population, preferably atleast 20% higher. Other components of the splenocyte population (e.g.macrophages, granulocytes, platelets) can be contained in either theT-cell fraction or the B-cell fraction. Methods to affect thefractionation of the splenocyte population will be evident to thoseskilled in the art, examples being rosetting with sheep red blood cells;rosetting with red blood cells coated with anti-T or anti-B cellantibodies; panning or separation of cells on solid matrix coated withunmodified antibodies or antibodies conjugated with labels (e.g.biotin); separation of cells labeled with antibodies conjugated withfluorochromes by a fluorescence-activated cell sorter;complement-mediated lysis of cells coated with appropriate antibodies;gradient separation (e.g. l-g sedimentation); electrophoresis; and thepreferred method for purposes of the instant invention-passing thesplenocyte population over a nylon wool separation column having a highsurface area to volume ratio (explained in more detail in the Examplebelow).

After fractionation of the splenocyte population, the T-cell fraction(first fraction) and the B-cell fraction (second fraction) arerecombined in a manner to form a second population having a T-helpercell to B-cell ratio of at least 0.4 while maintaining the percentage ofT-suppressor cells about the same as that of the original splenocytepopulation. By utilizing the nylon wool fractionation proceduredescribed in the Example below, such a T-helper cell/B-cell ratio, andT-suppressor cell percentage, is obtained. If fractionation is to becarried out by one of the other methods listed above, the ratio ofT-helper cells to B-cells, and the T-suppressor cell percentage, arecontrolled by such methods as will be apparent to those skilled in theart utilizing antibodies against helper T-cells as appropriate in thosemethods.

Following the fractionation and recombination steps, the secondpopulation is cultured (in appropriate tissue culture media such asRPMI-1640, Dulbecco's Modified Eagles' Media, or Iscove's ModifiedDulbecco's Media) in the presence of human serum (allogeneic ispreferred, although autologous may be utilized), a lymphokine(s) capableof inducing proliferation and differentiation of T and B cells,optionally a monocyte activator, and a clinically relevant immunogen.

The lymphokines useful in the practice of the subject invention will beapparent to those skilled in the art and include IL-1 and IL-2, B-cellgrowth factor, B-cell differentiation factor, interferon,colony-stimulating factor, thymic hormones, maturation factor andepidermal growth factor. Monocyte activators useful in the practice ofthe subject invention include phorbol esters, muramyl dipeptide,complement components, microbial constituents (e.g. endotoxin)interferon and interferon inducers (e.g. polyanions and viruses),mitogens, Sepharose beads and protozoa.

The selection of an immunogen against which antibodies are to be raisedwill, of course, depend upon clinical interest. Some clinicallysignificant immunogens include bacterial antigens, viral antigens,toxins, blood group antigens, antigens on lymphoid cells, myosin, andtumor antigens such as cell-associated antigens and tumor cell secretedproducts (e.g., bombesin, CEA, AFP, HCG, calcitonin, ACTH, AVP andneurotensin). As is well known in the art, smaller antigens (mol. wt. ofless than about 5000) may need to be coupled to a carrier in order tostimulate an effective immune response against the immunogen.Preferably, this carrier is an entity against which the humanlymphocytes have been previously immunized, e.g. tetanus toxoid.Examples of useful carriers are keyhole-limpet hemocyanin,thyroglobulin, albumins, muramylxdipeptide, red blood cells, a solidmatrix such as Sepharose beads, alkaline phosphatase, globulins,synthetic copolymers, fibrinogen and the like. Some smaller antigens mayalso be polymerized to increase immunogenicity. Linking agents useful inthe coupling of smaller antigens to carriers include carbodiimides;glutaraldehyde; N-N-carbonyldiimidazole; 1-hydroxybenzotriazolemonohydrate; N-hydroxy succinimide; N-trifluoroacetylimidazole; cyanogenbromide; and bis-diazotized benzidine. The concentration of the antigennecessary for purposes of the instant invention will depend upon thesize of the antigen, and will generally be in the range of 0.1 to100,000 ng/ml, preferably 1-10,000 ng/ml.

After culturing the splenocytes, cells are fused with an appropriatefusion partner cell line (e.g. NS-1, SHM D33, SBC H20, P3Ag8-653, SP2/0,HMy-2, WI-L2 729HF2, GM4672, and UC729-6) and the hybridoma supernatantscreened for antibody production. Alternatively, cells are immortalizedby transformation techniques using EBV, oncogenic DNA, etc.

Antibodies produced in accordance with the immunization protocol of theinstant invention are useful in any of the well known diagnostic ortherapeutic techniques currently utilizing monoclonal antibodies. Theantibodies can be labeled with enzymes to be used in various enzymelinked immunoassays, with radiolabels to be used in RIAs or in in-vivodiagnosis or therapeutics, and can be used unlabeled in variouscompetitive binding assays and therapeutic applications.

The instant invention is described in more detail in the followingExample, such Example is by no means intended to limit the scope of theinvention.

EXAMPLE 1. Materials and Methods

Purification of Tetanus Toxoid

Tetanus toxoid concentrate was purchased from Massachusetts State PublicHealth Laboratories and purified by fast protein liquid chromatography(FPLC). Typically, 5 ml of concentrate (containing 10 mg specificproteins) were applied to a 0.5×5 cm Mono Q resin column (Pharmacia,Piscataway, N.J.) which was previously equilibrated with 50 mM Bis/Tris,pH8.0. Elution was performed using a stepwise gradient with 50 mMBis/Tris, 1M NaCl, pH8.0 as limit buffer. Greater than 95% of thetetanus toxid was recovered at 40% of the limit buffer and dialysedagainst 0.15M NaCl. The purity of tetanus toxoid was monitored bySDS-PAGE and reactivity with human antibodies against tetanus toxoid(Hepertet, Cutter Biological, Berkeley, CA).

Conjugation of [Lys³ ]-Bombesin to Protein Carriers

[Lys³ ]-bombesin (M.W. 1592), (Bachem, Burlingame, CA) was conjugated toFPLC-purified tetanus toxoid (M.W. 180,000) via 1 ethyl3-(3-dimethyl-aminopropyl) carodiimide hydrochloride (ECDI) (Calbiochem,San Diego, CA) according to a modified procedure of Goodfriend et. al.(cite). The molar ratio of bombesin:carrier: ECDI was generally30-60:1:6900. Briefly, [Lys³ ]-bombesin at 2 mg/ml was added dropwise totetanus toxoid in 0.15M NaCl followed by the dropwise addition of 200 ulof ECDI with swirling. After incubating for 15-30 minutes at roomtemperature, the mixture was applied to a G75 column to remove theunconjugated bombesin. Fractions containing bombesin-protein conjugateswere pooled and stored at -20° C. Efficiency of conjugation wasmonitored by immunoreactivity with rabbit anti-bombesin (gift of Dr. L.H. Lazarus, National Institute of Environmental Health Sciences) inELISA and by amino acid analysis.

Lymphocyte Culture Supernatants (LCS)

LCS were kindly provided by Dr. James Woody (Naval Research Center,Bethesda, MD). Briefly, they were prepared as follows. Peripheral bloodlymphocytes from screened donors were cultured at 1×10⁶ /ml in RPMI-1640medium supplemented with 0.1% purified PHA-P (Difco Laboratories,Detroit, MI) and 5% human AB serum; for 48 hours. The culturesupernatants were harvested, filtered through 0.22 μm filters, andassays for interleukin-2 (IL-2) activity on an IL-2 dependent humanT-cell clone. Lots with >0.8 U/ml of IL-2 were pooled and stored at 4°C. before use. Human BCGF activity was also detected in thesesupernatants by using a Staphylococcus aureus costimulator assay.

Immunofluorescent Staining

Two million human lymphocytes were incubated with 1 μg of mousemonoclonal antibody 64.1 (anti-B cells), 2H7 (anti-T cells), 66.1(anti-helper/inducer T cells), or 51.1 (anti-cytotoxic/suppressor Tcells) (DuPont-NEN Products, Boston, MA) in 100 μl of Hanks BalancedSalt Solution (GIBCO, Grand Island, N.Y.) supplemented with 2% FCS and0.1% sodium azide for 30 minutes at 4° C. The cells were then washedthree times and 100 μl of FITC-conjugated sheep F(ab')₂ anti-mouse lg(DuPont-NEN) diluted 1:100 were added. After another incubation of 30minutes at 4° C., the cells were washed three times, resuspended inphosphate-buffered saline (PBS) with 1% formalin and analysed on anEPICS V flow cytometer (Coulter, Hialeah, FL).

Enzyme-linked Immunoassays (ELISA)

Ninety-six well Immulon II plates (Dynatech, Alexandria, VA) were coatedwith 400 ng per well of bombesin conjugated to protein carriers of freeprotein carriers diluted in carbonate buffer, pH 9.6 for 18 hr. at 4° C.The plates were washed with PBS, pH 7.2, three times and blocked withPBS-1% bovine serum albumin (BSA) for 1 hour at 37° C. Subsequently, theplates were washed three times with PBS and 50 μl of hybrid supernatantfluids were added per well. After incubating for 1 hours at 37° C., theplates were again washed three times with PBS and 50 μl of horseradishperoxidase (HRP) conjugated goat anti-human lg (Cooper Biomedical,Malvern, PA), were added per well. The plates were then incubated at 37°C. for 1 hour, washed three times with PBS, and the enzyme detected bythe addition of 100 μl of o-phenylene diamine and 0.15% hydrogenperoxide. All antibodies were diluted in PBS with 0.05% Tween 20. Insome experiments, HRP goat anti-human lg was substituted by goatanti-human lgM or goat anti-human lgG (Cooper Biomedical) for isotypedetermination of the specific antibodies.

Competitive Inhibition Studies

Antibodies to be used in competitive inhibition studies were firsttitrated on plates coated with bombesin-tetanus toxoid conjugates (BTT)to determine the concentration of antibody giving half-maximal binding.This half-maximal concentration of antibody was preincubated withvarious concentrations of soluble inhibitors at 37° C. for 3 hours. Theinhibitors included bombesin conjugated to tetanus toxoid orthyroglobulin via ECDl, tetanus toxoid, thyroglobulin, bombesin coupledto thyroglobulin by glutaraldehyde, and free [Lys³ ]-bombesin. Afterpreincubation, the antibodies were added to plates coated with BTT andincubated for 15 minutes at 37° C. The plates were then processed asdescribed for routine ELISA.

In vitro Immunization

Single cell suspensions of splenocytes were prepared by teasing splenictissues in RPMI-1640 medium (M.A. Bioproducts, Bethesda, Md.). The redcells were removed by centrifugation on a gradient oflymphocyte-separation medium (Litton Bionetics, Rockville, Md.) at1400×g for 20 minutes. Cells recovered from the interfaces were washedthree times in RPMI-1640 and resuspended at 10⁷ cells/ml in ice-coldRPMI-1640 medium supplemented with 40% FCS and 10% dimethylsulfoxide.Subsequently, the cells were frozen in a controlled rate-freezer(CryoMed, St. Clemens, Mich.).

For sensitization, cells were thawed quickly at 37° C. and washed twotimes with RPMI-1640 medium. Spleen cells (1.5-2×10⁷) were loaded ontocolumns made of 0.9 g nylon wool in a 20 ml syringe barrel. Thenonadherent cells were collected by elution whereas the adherent cellswere harvested by alternatively washing and squeezing the nylon woolwith a syringe plunger. The separated cells were washed two times inRPMI-1640 medium. One million cells of each population were added toeach 16 mm culture well with antigen, 10% human AB serum (Biobee,Boston, Mass.), 7.5 μg/ml E coli lipopolysaccharide (Difco, Detroit,Mich.), and 20% pooled PHA activated lymphocyte culture supernatants(LCS). The cells in a final volume of 2 ml were cultured at 37° C. in10% CO₂ for 4-6 days. The antigen was soluble [Lys³ ]-bombesin coupledto tetanus toxoid. Typically, 3 or more concentrations of antigens wereused in each experiment and 8-10 wells were set up for eachconcentration.

At the end of the sensitization period, the cells from identical wellswere pooled and washed two times with RPMI-1640 medium. They were thenfused with equal numbers of NS-1 cells using 50% polyethylene glycol(PEG, M.W. 1000) or 45% PEG (M.W. 4000) utilizing conventional protocol.The fused cells were plated at 10⁵ lymphocytes/well in RPMI-1640supplemented with 20% FCS, 2 mM glutamine, 1 mM sodium pyruvate, 5%NCTC-109 (GIBCO, Grand Island, NY), 10 μg/ml gentamycin, 10⁻⁴ Mhypoxanthine, 4×10⁻⁷ M aminopterin, and 1.6×10⁻⁵ M thymidine. Allogeneicirradiated (2000 R) peripheral blood lymphocytes were added at 1.5×10⁴/well as feeder cells. Hybrids appeared in 2-3 weeks and were screenedfor anti-bombesin antibodies by ELISA.

II. Results

Immunofluorescent studies on the two cell populations separated on nylonwool showed that the nonadherent fraction was enriched in T lymphocytes(77% in nonadherent fraction vs. 37% in original population) whereas theadherent fraction was enriched in B lymphocytes (83% in adherentfraction vs. 64% in original population).

The two populations were mixed in equal numbers which resulted in asecond population having a T-helper cell to B-cell ratio of about 0.63,and a T-suppressor cell content about equal to that of the originalpopulation. This second population was cultured with 20% LCS, 10% humanAB serum, 7.5 μg/ml LPS, and soluble BTT. Because the concentration ofantigen giving optimal stimulation varied among donors, 3 to 4 doses ofBTT ranging from 0.6 ng/ml-6 μg/ml were tested in each experiment. After6 days of culture, the sensitized cells were fused with NS-1 cells andthe hybrids assayed for reactivity with BTT, BTG, TT or TG 3-4 weeksafter fusion. A hybrid (BM-1) was selected that produced antibody whichbound to BTT and BTG but not TT nor TG. This reactivity pattern stronglysuggested the presence of an antibody against bombesin. The use ofisotype-specific HRP-conjugated antibodies in the ELISA showed that thespecific antibody was lgM. This hybrid was cloned by limiting dilutionand its specificity confirmed by titrations in ELISA.

The binding of antibody BM-1 to BTT and BTG, but not TT or TG, stronglysuggested that it was an anti-bombesin. However, to further study thespecificity of putative anti-bombesin antibodies, competitive inhibitionwas performed using a dilution of antibody which had been foundpreviously to yield half-maximal binding to BTT. Binding of the BM-1antibody to BTT was inhibited by precincubation of the hybridsupernatant fluids with bombesin conjugated to TT or TG via ECDI. Incontrast, unconjugated TT or TG was not inhibitory. This reactivitypattern confirms that observed in the initial ELISA. To rule out thepossibility that the antibody is recognizing a determination on thecoupling agent, ECDI, unconjugated [Lys³ ]-bombesin and BTG prepared byconjugation via glutaraldehyde were included as inhibitors. Bothreagents were inhibitory indicating that BM-1 was producing a genuineanti-bombesin antibody.

I claim:
 1. A process for the in vitro immunization of an immuno-competent human splenocyte against an immunogen comprising:(a) obtaining a first human splenocyte population; (b) fractionating the first population by contacting with nylon wool so that a first fraction is enriched with T-cells and a second fraction is enriched with B-cells, wherein said T-cells have subpopulations of T-helper cells and T-suppressor cells; (c) mixing together approximately equal numbers of cells from the first and second fractions to form a second population such that the second population has a T-helper cell to B-cell ratio of at least 0.4 and such that the percentage of T-suppressor cells is essentially unchanged from the first population; and (d) culturing the second population in a medium containing human serum, an immunogen, and a lymphokine or lymphokines which induce proliferation and differentiation of T and B cells wherein the human serum, the immunogen and the lymphokine or lymphokines are added at initiation of culture.
 2. The process of claim 1 wherein step (b) comprises contacting the first B-cell enriched population with nylon wool whereby the second fraction is adsorbed to the nylon wool and the first fraction does not adsorb to nylon wool, the second fraction being subsequently desorbed for use in step (c).
 3. The process of claim 1 or 2 wherein "the medium " of step (d) further contains a "monocyte activator".
 4. The process of claim 1 or 2 wherein the human serum is "allogeneic".
 5. The process of claim 3 wherein the human serum is allogeneic.
 6. The process of claim 1 or 2 wherein the immunogen is selected from the group consisting of bacterial antigens, viral antigens, toxins, blood group antigens, lymphoid cell antigens, myosin and tumor antigens.
 7. The process of claim 1 or 2 wherein the immunogen is a tumor cell secreted product.
 8. The process of claim 6 wherein "the medium" of step (d) further contains a "monocyte activator".
 9. The process of claim 6 wherein the human serum is allogeneic. 